■NPR1同源基因全长cDNA的分离与表达分析
|
摘要
NPR1是水杨酸诱导抗病基因表达的重要调控因子之一,主要作用于水杨酸信号转导途径的上、下游。采用稀释池PCR法,筛选■(Prunus salicina var. cordata)叶片全长均一化cDNA文库,分离■叶片NPR1同源基因的全长cDNA;用不同浓度的水杨酸喷施一年生■嫁接苗嫩叶,采用荧光定量PCR技术检测所获得的NPR1同源基因的表达量差异。试验结果表明,共获得了4个NPR1同源基因的全长cDNA,分别命名为PsNPR1、PsNPR3.1、PsNPR3.2和PsNPR3.3,其长度分别为2 442、1 827、2 244和2 615 bp,其ORF长度分别为1 788、1 770、1 767和1 782 bp,分别编码596、590、589和594个氨基酸的蛋白质;氨基酸序列比对结果表明,■这4个NPR1同源基因的编码区均含有BTB/POZ和ANK两个保守域及核定位信号(NLS);PsNPR1与拟南芥的AtNPR1和At NPR2聚为一簇,PsNPR3.1、PsNPR3.2和PsNPR3.3与拟南芥的AtNPR3和At NPR4聚为另一簇。PsNPR1在喷施1.0 mmol·L~(-1)水杨酸的叶片中表达量最高;PsNPR1、PsNPR3.1、PsNPR3.2在响应1.0 mmol·L~(-1)水杨酸诱导过程中表达量持续上升,30h表达量最高,之后呈下降趋势,因此,水杨酸诱导抗病基因高表达的最佳处理浓度为1.0 mmol·L~(-1),最佳响应时间为30 h。
NPR1 is one of important regulatory factors inducing expression of desease-resistant genes,and mainly acts in the up-or down-streams on salicylic acid signal transduction pathway. Full-length cDNAs of NPR1 homologous genes were screened from enriched full-length normalized cDNA library by pool-dilued PCR method in Nai's leaves;young leaves in one-year grafted seedlings were sprayed with salicylic acid solutions of different concentrations,the differences in expression levels of the isolated NPR1 genes were detected by fluorescence quantitative PCR. The results showed that 4 full-length cDNAs of NPR1 homologous genes were obtained and named as PsNPR1,PsNPR3.1,PsNPR3.2 and PsNPR3.3 respectively;their responding full lengths were 2 442,1 827,2 244 and 2 615 bp;their reponding lengths of ORFs were 1 788,1 770,1 767 and 1 782 bp,encoding proteins containing 596,590,589 and 594 amino acids,respectively;the results of amino acid sequence alignment showed that coding regions of the 4 NPR1 homologous genes contained two conservative domains(BTB/POZ and ANK)and nuclear localization signal(NLS);Nai's PsNPR1 was clustered with Arabidopsis thaliana's AtNPR1 and AtNPR2 forming a cluster,Nai's PsNPR3.1,PsNPR3.2 and PsNPR3.3 were clustered with Arabidopsis thaliana's AtNPR3 and AtNPR4 forming another cluster. The expression level of PsNPR1 was the highest in Nai's young leaves sprayed with 1.0 mmol · L~(-1) salicylic acid solutions;the expression levels of PsNPR1,PsNPR3.1 and PsNPR3.2 continuously increased in process of response to 1.0 mmol · L~(-1) salicylic acid,and reached maximum at 30 hours after spraying,then tended to decrease,therefore,the optimal spraying concentration of salicylic acid was 1.0 mmol · L~(-1),the optimal responsing time of 1.0 mmol · L~(-1) salicylic acid was 30 hours.
NPR1 is one of important regulatory factors inducing expression of desease-resistant genes,and mainly acts in the up-or down-streams on salicylic acid signal transduction pathway. Full-length cDNAs of NPR1 homologous genes were screened from enriched full-length normalized cDNA library by pool-dilued PCR method in Nai's leaves;young leaves in one-year grafted seedlings were sprayed with salicylic acid solutions of different concentrations,the differences in expression levels of the isolated NPR1 genes were detected by fluorescence quantitative PCR. The results showed that 4 full-length cDNAs of NPR1 homologous genes were obtained and named as PsNPR1,PsNPR3.1,PsNPR3.2 and PsNPR3.3 respectively;their responding full lengths were 2 442,1 827,2 244 and 2 615 bp;their reponding lengths of ORFs were 1 788,1 770,1 767 and 1 782 bp,encoding proteins containing 596,590,589 and 594 amino acids,respectively;the results of amino acid sequence alignment showed that coding regions of the 4 NPR1 homologous genes contained two conservative domains(BTB/POZ and ANK)and nuclear localization signal(NLS);Nai's PsNPR1 was clustered with Arabidopsis thaliana's AtNPR1 and AtNPR2 forming a cluster,Nai's PsNPR3.1,PsNPR3.2 and PsNPR3.3 were clustered with Arabidopsis thaliana's AtNPR3 and AtNPR4 forming another cluster. The expression level of PsNPR1 was the highest in Nai's young leaves sprayed with 1.0 mmol · L~(-1) salicylic acid solutions;the expression levels of PsNPR1,PsNPR3.1 and PsNPR3.2 continuously increased in process of response to 1.0 mmol · L~(-1) salicylic acid,and reached maximum at 30 hours after spraying,then tended to decrease,therefore,the optimal spraying concentration of salicylic acid was 1.0 mmol · L~(-1),the optimal responsing time of 1.0 mmol · L~(-1) salicylic acid was 30 hours.
引文
Armou J A L. 2006. Tandemly repeated DNA:why should anyone care? Mutation Research, 598(1-2):6-14.
Cao H, Glazebrook J, Clarke J D, Volko S, Dong X. 1997. The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell, 88(1):57-63.
Chen X K,Zhang J Y,Zhang Z,Du X L,Du B B, Qu S C. 2012. Overexpressing MhNPRl in transgenic Fuji apples enhances resistance to apple powdery mildew. Molecular Biology Reports, 39(8):8083-8089.
Coqueiro D S O,de Souza A A,Takita M A,Rodrigues C M, Kishi L T, Machado M A. 2015. Transcriptional profile of sweet orange in response to chitosan and salicylic acid. BMC Genomics, 16(1):288.
Deng-wei J, Liu Y, Ce S, Min C, Qing Y. 2014. Cloning and characterization of a Solanum torvum NPR1 gene involved in regulating plant resistance to Verticillium dahliae. Acta Physiologiae. Plantarum, 36(11):2999-3011.
Dong Chunjuan, Cao Ning,Shang Qingmao. 2017. Effects of salicylic acid on fatty acid compositions in the roots of cucumber seedlings under low temperature. Acta Horticulturae Sinica, 44(7):1319-1326.(in Chinese)董春娟,曹宁,尚庆茂.2017.外源水杨酸对低温胁迫下黄瓜幼苗根系脂肪酸不饱和度的影响.园艺学报,44(7):1319-1326.
Fu Z Q, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel S H, Tada Y, Zheng N, Dong X. 2012. NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature, 486(7402):3153.
Gao Lijuan,Zhang Yuxing. 2013. Effects of salicylic acid on the expression of SOD, PPO isozymes and NPR1 in pear. Acta Horticulturae Sinica,40(1):41-48.(in Chinese)高丽娟,张玉星.2013.水杨酸对梨SOD、PPO同工酶和NPR1表达的影响.园艺学报,40(1):41-48.
Lu Ying,Zhang Xin,Qi Yanxiang,Pu Jinji, Xie Yixian. 2010. Clonging and construction of a plant expressing vector of NPR1 gene from banana.Chinese Journal of Tropical Crops, 31(9):1474-1479.(in Chinese)陆英,张欣,漆艳香,蒲金基,谢艺贤.2010.香蕉NPR1基因的克隆及植物表达载体构建.热带作物学报,31(9):1474-1479.
Moreau M, Tian M, Klessig D F. 2012. Salicylic acid binds NPR3 and NPR4 to regulate NPR1-dependent defense responses. Cell Research, 22(12):1631-1633.
Mou Z,Fan W,Dong X. 2003. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell, 113(7):935-944.
Queval G,Issakidis-Bourguet E, Hoeberichts F A, Vandorpe M, Gakiere B, Vanacker H, Miginiac-Maslow M, Van Breusegem F, Noctor G.2007. Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel. The Plant Journal, 52(4):640-657.
Rochon A, Boyle P, Wignes T, Fobert P R, Despr E S C. 2006. The coactivator function of Arabidopsis NPR1 requires the core of its BTB/POZ domain and the oxidation of C-terminal cysteines. The Plant Cell, 18(12):3670-3685.
Spoel S H, Mou Z, Tad Y, Spivey N W, Genschik P, Dong X. 2009. Proteaso me-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell, 137(5):860-872.
Spoel S H,Dong X. 2012. How do plants achieve immunity? Defence without specialized immune cells. Nature Reviews Immunology, 12(2):89-100.
Vidhyasekaran P. 2015. Salicylic acid signaling in plant innate immunity. Berlin:Springer Netherlands:27-122.
Wu Q,Wang X Z,Tang Y Y,Yu H T,Ding Y F,De Yang C,Cui F G,Zhang J C,Wang C T. 2014. Molecular cloning and characterization of NPR1 gene from Arachis hypogaea. Molecular Biology Reports, 41(8):5247-5256.
Yang Ye. 2013. Cloning and expression of MYB genes in Narcissus tazetta var. chinesis[M. D. Dissertation]. Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)杨晔.2013.中国水仙MYB转录因子基因的克隆与表达分析[硕士论文].福州:福建农林大学.
Zhang Jiyu. 2011. Clonging and function analysis of disease resistance genes of Malus Hupehensis[Ph. D. Dissertation]. Nanjing:Nanjing Agricultural University.(in Chinese)张计育.2011.湖北海棠抗病相关基因的克隆及其功能分析[博士论文].南京:南京农业大学.
Zhang Y, Fan W, Kinkema M, Li X, Dong X. 1999. Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proceedings of the National Academy of Science, 96(11):6523-6528.
Zhang Y,Tessaro M J,Lassner M, Li X. 2003. Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistance. The Plant Cell, 15(11):2647-2653.
Zhao Li. 2014. Isolation and expression of full-length cdnas of genes encoding key enzymes in process of formation of waxes on nai's buds and leaves[M. D. Dissertation]. Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)赵利.2014.栋芽叶蜡质形成关键酶基斟因的分离与表达研究[硕士论文].福州:福建农林大学.
Zhong X, Xi L, Lian Q, Luo X, Wu Z, Seng S, Yuan X, Yi M. 2015. The NPR1 homolog GhNPRl plays an important role in the defense response of Gladiolus hybridus. Plant Cell Reports, 34(6):1063-1074.
Cao H, Glazebrook J, Clarke J D, Volko S, Dong X. 1997. The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell, 88(1):57-63.
Chen X K,Zhang J Y,Zhang Z,Du X L,Du B B, Qu S C. 2012. Overexpressing MhNPRl in transgenic Fuji apples enhances resistance to apple powdery mildew. Molecular Biology Reports, 39(8):8083-8089.
Coqueiro D S O,de Souza A A,Takita M A,Rodrigues C M, Kishi L T, Machado M A. 2015. Transcriptional profile of sweet orange in response to chitosan and salicylic acid. BMC Genomics, 16(1):288.
Deng-wei J, Liu Y, Ce S, Min C, Qing Y. 2014. Cloning and characterization of a Solanum torvum NPR1 gene involved in regulating plant resistance to Verticillium dahliae. Acta Physiologiae. Plantarum, 36(11):2999-3011.
Dong Chunjuan, Cao Ning,Shang Qingmao. 2017. Effects of salicylic acid on fatty acid compositions in the roots of cucumber seedlings under low temperature. Acta Horticulturae Sinica, 44(7):1319-1326.(in Chinese)董春娟,曹宁,尚庆茂.2017.外源水杨酸对低温胁迫下黄瓜幼苗根系脂肪酸不饱和度的影响.园艺学报,44(7):1319-1326.
Fu Z Q, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel S H, Tada Y, Zheng N, Dong X. 2012. NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature, 486(7402):3153.
Gao Lijuan,Zhang Yuxing. 2013. Effects of salicylic acid on the expression of SOD, PPO isozymes and NPR1 in pear. Acta Horticulturae Sinica,40(1):41-48.(in Chinese)高丽娟,张玉星.2013.水杨酸对梨SOD、PPO同工酶和NPR1表达的影响.园艺学报,40(1):41-48.
Lu Ying,Zhang Xin,Qi Yanxiang,Pu Jinji, Xie Yixian. 2010. Clonging and construction of a plant expressing vector of NPR1 gene from banana.Chinese Journal of Tropical Crops, 31(9):1474-1479.(in Chinese)陆英,张欣,漆艳香,蒲金基,谢艺贤.2010.香蕉NPR1基因的克隆及植物表达载体构建.热带作物学报,31(9):1474-1479.
Moreau M, Tian M, Klessig D F. 2012. Salicylic acid binds NPR3 and NPR4 to regulate NPR1-dependent defense responses. Cell Research, 22(12):1631-1633.
Mou Z,Fan W,Dong X. 2003. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell, 113(7):935-944.
Queval G,Issakidis-Bourguet E, Hoeberichts F A, Vandorpe M, Gakiere B, Vanacker H, Miginiac-Maslow M, Van Breusegem F, Noctor G.2007. Conditional oxidative stress responses in the Arabidopsis photorespiratory mutant cat2 demonstrate that redox state is a key modulator of daylength-dependent gene expression, and define photoperiod as a crucial factor in the regulation of H2O2-induced cel. The Plant Journal, 52(4):640-657.
Rochon A, Boyle P, Wignes T, Fobert P R, Despr E S C. 2006. The coactivator function of Arabidopsis NPR1 requires the core of its BTB/POZ domain and the oxidation of C-terminal cysteines. The Plant Cell, 18(12):3670-3685.
Spoel S H, Mou Z, Tad Y, Spivey N W, Genschik P, Dong X. 2009. Proteaso me-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell, 137(5):860-872.
Spoel S H,Dong X. 2012. How do plants achieve immunity? Defence without specialized immune cells. Nature Reviews Immunology, 12(2):89-100.
Vidhyasekaran P. 2015. Salicylic acid signaling in plant innate immunity. Berlin:Springer Netherlands:27-122.
Wu Q,Wang X Z,Tang Y Y,Yu H T,Ding Y F,De Yang C,Cui F G,Zhang J C,Wang C T. 2014. Molecular cloning and characterization of NPR1 gene from Arachis hypogaea. Molecular Biology Reports, 41(8):5247-5256.
Yang Ye. 2013. Cloning and expression of MYB genes in Narcissus tazetta var. chinesis[M. D. Dissertation]. Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)杨晔.2013.中国水仙MYB转录因子基因的克隆与表达分析[硕士论文].福州:福建农林大学.
Zhang Jiyu. 2011. Clonging and function analysis of disease resistance genes of Malus Hupehensis[Ph. D. Dissertation]. Nanjing:Nanjing Agricultural University.(in Chinese)张计育.2011.湖北海棠抗病相关基因的克隆及其功能分析[博士论文].南京:南京农业大学.
Zhang Y, Fan W, Kinkema M, Li X, Dong X. 1999. Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proceedings of the National Academy of Science, 96(11):6523-6528.
Zhang Y,Tessaro M J,Lassner M, Li X. 2003. Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistance. The Plant Cell, 15(11):2647-2653.
Zhao Li. 2014. Isolation and expression of full-length cdnas of genes encoding key enzymes in process of formation of waxes on nai's buds and leaves[M. D. Dissertation]. Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)赵利.2014.栋芽叶蜡质形成关键酶基斟因的分离与表达研究[硕士论文].福州:福建农林大学.
Zhong X, Xi L, Lian Q, Luo X, Wu Z, Seng S, Yuan X, Yi M. 2015. The NPR1 homolog GhNPRl plays an important role in the defense response of Gladiolus hybridus. Plant Cell Reports, 34(6):1063-1074.